Our senses of hearing and balance rely on the proper development, function and maintenance of the hair bundle at the apical surface of inner ear hair cells. Hair bundles are composed of distinct multi-protein complexes of defined function and architecture that work together and that are responsible for mechanoelectrical transduction, adaptation and possibly signal amplification. Failure of function of any of these machineries leads to deafness or various degrees of hearing and balance impairments. Our goal is to visualize these stereocilia molecular machines in their native cellular context at molecular resolution. Our rationale is that the determination of the molecular 3D organization of these stereocilia complexes will ultimately lead to a mechanistic understanding of development, function and maintenance of this precision organelle. Specifically, we propose specifically to study four regions in stereocilia from lower (frog, zebrafish) and higher (mouse, guinea pig) vertebrates, each of which hosts a particular type of multi-protein complex, and plays a distinct role in hair bundle formation, function and/or maintenance. The four regions are 1.) the tip complex, 2.) the actin core, 3.) the adaptation complex, as well as 4.) the tapered region and the rootlets. Our specific aims are as follows: Specific Aim 1: Determine 3D organization of sterecilia tip complex, the site of mechanotransduction and actin bundle nucleation Specific Aim 2: Determine 3D organization of side plaque, the site of adaptation and possibly signal amplification. Specific Aim 3: Determine 3D organization of the actin core, including the actin-actin cross linkers and the actin bundle-membrane connectors. Specific Aim 4: Determine the 3D organization of molecular machines residing in the tapered stereocilia region, the rootlets and the cuticular plate We will employ electron tomography of either high-pressure frozen, freeze-substituted and resin-embedded hair bundle sections or of frozen-hydrated isolated individual stereocilia. The 3D architecture of the bundle multiprotein complexes will aid in a mechanistic understanding of our senses or hearing and balance and provide a baseline for future analysis of diseased tissue.